Abstract The hemoglobin switch -- repression of ?-globin and activation of ?-globin expression during the transition from fetal to adult stages of erythropoiesis -- is a major paradigm for cell and developmental stage specific regulation. Given family and genetic studies demonstrating the beneficial effects of increased HbF (?2?2) in the ?-hemoglobin disorders -- sickle cell disease (SCD) and ?-thalassemia -- efforts have been directed towards manipulating the switch as a therapeutic approach. A major obstacle, however, has been ignorance regarding how the switch is achieved and ?-globin silencing is maintained in the adult. Work in our group over the past several years has established the repressor protein BCL11A as a central, quantitative regulator of the switch. Moreover, critical sequences in an erythroid-specific enhancer within the BCL11A gene are required for its expression, providing a target site for gene editing as an improved form of gene therapy for patients with ?-hemoglobin disorders. The remaining challenge now is development of therapeutic strategies based on modulation of BCL11A function with small molecules, as a means to bring mechanism-based therapy to wider populations. In this project we will focus on the BCL11A protein and address three aspects. First, following our identification of components of the nuclear matrix in association with BCL11A protein complexes, we will dissect the functional relationship between chromatin and the nuclear matrix in erythroid cells. Second, with the goal of reducing the level of BCL11A protein and relieving HbF repression, we will employ genetic and small molecule screens to test the possibility that inhibition of deubiquitylases might constitute a new therapeutic approach to HbF reactivation. Third, we will perform saturating CRISPR/Cas9 mutagenesis of the human A??? intergenic region to localize sequences required for full HbF repression, and correlate these with chromatin sites proposed to bind BCL11A. In this manner, we will identify critical cis-acting sequences bound by BCL11A, ascertain whether these are sites of chromatin-nuclear matrix interaction, and define additional DNA sequences for therapeutic genome editing. The goal of this project is to capitalize on our discovery of BCL11A as a major regulator of the hemoglobin switch and develop innovative, and non-traditional, therapeutic approaches to the hemoglobin disorders.